U.S. patent application number 12/618461 was filed with the patent office on 2010-03-11 for cytotoxin compound and method of isolation.
This patent application is currently assigned to University of South Florida. Invention is credited to Charles D. Amsler, Bill J. Baker, Thushara Diyabalanage, James B. McClintock.
Application Number | 20100063137 12/618461 |
Document ID | / |
Family ID | 34885918 |
Filed Date | 2010-03-11 |
United States Patent
Application |
20100063137 |
Kind Code |
A1 |
Baker; Bill J. ; et
al. |
March 11, 2010 |
Cytotoxin Compound And Method of Isolation
Abstract
The present invention concerns compounds derived from tunicates
of the species Synoicum adareanum, as well as to pharmaceutical
compositions comprising these compounds and methods of use.
Extracts from tunicates show selective toxicity against several
different cancer cell lines in the NCI 60 cell line panel. These
compounds are useful in the effective treatment of cancers,
particularly malignant melanomas, colon cancer, and renal cancer
cell lines.
Inventors: |
Baker; Bill J.; (Tampa,
FL) ; Diyabalanage; Thushara; (Tampa, FL) ;
McClintock; James B.; (Birmingham, AL) ; Amsler;
Charles D.; (Pelham, AL) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO Box 142950
GAINESVILLE
FL
32614
US
|
Assignee: |
University of South Florida
Tampa
FL
The UAB Research Foundation
Birmingham
AL
|
Family ID: |
34885918 |
Appl. No.: |
12/618461 |
Filed: |
November 13, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10906386 |
Feb 17, 2005 |
7625885 |
|
|
12618461 |
|
|
|
|
60521073 |
Feb 17, 2004 |
|
|
|
Current U.S.
Class: |
514/450 ;
549/271 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/365 20130101; C07D 313/00 20130101 |
Class at
Publication: |
514/450 ;
549/271 |
International
Class: |
A61K 31/365 20060101
A61K031/365; C07D 313/00 20060101 C07D313/00; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was developed under support from the National
Science Foundation under grants OPP-9901076 and OPP-0125152;
accordingly the U.S. government has certain rights in the
invention.
Claims
1. A method of treating a subject with cancer or a cell
proliferation disorder, comprising administering to the subject a
therapeutically effective amount of at least one isolated compound
obtained from extracts of a Synoicum species, or a composition
comprising said compound.
2. The method of claim 1, wherein said Synoicum species is S.
adareanum.
3. The method of claim 1, wherein said isolated compound obtained
from the Synoicum species is a Palmerolide.
4. The method of claim 3, wherein said Palmerolide comprises the
formula: ##STR00009## or a pharmaceutically acceptable salt,
isomer, racemate, or racemic mixture thereof.
5. The method of claim 4, wherein said salt is an acid salt of
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide,
hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
thiocyanate, tosylate or undecanoate.
6. The method of claim 4, wherein said salt is a base salt of
ammonium salts, alkali metal salts, alkaline earth metal salts,
salts with organic bases, N-methyl-D-glucamine, or salts with amino
acids.
7. The method of claim 3, wherein said Palmerolide comprises the
formula: ##STR00010## or a pharmaceutically acceptable salt,
isomer, racemate, or racemic mixture thereof.
8. The method of claim 7, wherein said salt is an acid salt of
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide,
hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
thiocyanate, tosylate or undecanoate.
9. The method of claim 7, wherein said salt is a base salt of
ammonium salts, alkali metal salts, alkaline earth metal salts,
salts with organic bases, N-methyl-D-glucamine, or salts with amino
acids.
10. The method of claim 3, wherein said Palmerolide comprises the
formula: ##STR00011## or a pharmaceutically acceptable salt,
isomer, racemate, or racemic mixture thereof.
11. The method of claim 10, wherein said salt is an acid salt of
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide,
hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
thiocyanate, tosylate or undecanoate.
12. The method of claim 10, wherein said salt is a base salt of
ammonium salts, alkali metal salts, alkaline earth metal salts,
salts with organic bases, N-methyl-D-glucamine, or salts with amino
acids.
13. The method of claim 3, wherein said Palmerolide comprises the
formula: ##STR00012## or a pharmaceutically acceptable salt,
isomer, racemate, or racemic mixture thereof.
14. The method of claim 13, wherein said salt is an acid salt of
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide,
hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
thiocyanate, tosylate or undecanoate.
15. The method of claim 13, wherein said salt is a base salt of
ammonium salts, alkali metal salts, alkaline earth metal salts,
salts with organic bases, N-methyl-D-glucamine, or salts with amino
acids.
16. The method of claim 1, wherein the cancer is melanoma, colon
cancer, or renal cancer.
17. The method of claim 1, wherein said composition comprises a
pharmaceutically acceptable carrier.
18. The method of claim 17, wherein said pharmaceutically
acceptable carrier comprises one or more of a diluent, excipient,
wetting agent, buffering agent, suspending agent, lubricating
agent, adjuvant, vehicle, delivery system, emulsifier,
disintegrant, absorbent, preservative, surfactant, colorant,
flavorant, or sweetener.
19. The method of claim 1, wherein said compound or composition is
administered to the subject parenterally, intravenously,
intradermally, subcutaneously, orally, transdermally, topically,
transmucosally, rectally, or by inhalation.
20. The method of claim 1, wherein said compound or composition is
provided as a controlled release formulation.
21. A composition comprising an isolated compound obtained from
extracts of a Synoicum species.
22. The composition according to claim 21, wherein said compound
comprises the formula: ##STR00013## or a pharmaceutically
acceptable salt, isomer, racemate, or racemic mixture thereof.
23. The composition according to claim 21, wherein said compound
comprises the formula: ##STR00014## or a pharmaceutically
acceptable salt, isomer, racemate, or racemic mixture thereof.
24. The composition according to claim 21, wherein said compound
comprises the formula: ##STR00015## or a pharmaceutically
acceptable salt, isomer, racemate, or racemic mixture thereof.
25. The composition according to claim 21, wherein said compound
comprises the formula: ##STR00016## or a pharmaceutically
acceptable salt, isomer, racemate, or racemic mixture thereof.
26. A method for inhibiting the proliferation of a cell, comprising
contacting said cell with an effective amount of an isolated
compound obtained from extracts of a Synoicum species, or a
composition comprising said compound.
27. The method of claim 26, wherein said isolated compound is a
Palmerolide.
28. The method of claim 26, wherein said compound comprises the
formula: ##STR00017## or a pharmaceutically acceptable salt,
isomer, racemate, or racemic mixture thereof.
29. The method of claim 26, wherein said compound comprises the
formula: ##STR00018## or a pharmaceutically acceptable salt, isomer
racemate, or racemic mixture thereof.
30. The method of claim 26, wherein said compound comprises the
formula: ##STR00019## or a pharmaceutically acceptable salt,
isomer, racemate, or racemic mixture thereof.
31. The method of claim 26, wherein said compound comprises the
formula: ##STR00020## or a pharmaceutically acceptable salt,
isomer, racemate, or racemic mixture thereof.
32. The method of claim 26, wherein said cell is a cancer cell.
33. The method of claim 32, wherein said cancer cell is a melanoma
cell, a colon cancer cell, or renal cancer cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 10/906,386, filed Feb. 17, 2005, which claims the benefit
of U.S. Provisional Application Ser. No. 60/521,073, filed Feb. 17,
2004, each of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] One of the greatest efforts of modern medicine is the
control and abatement of cellular proliferative disorders, such as
cancers. Considerable research has been conducted searching for new
biologically active compounds having useful activity for specific
cancers and the organisms which produce these compounds. For
example, certain marine soft corals have shown to be a source of
biologically active cytotoxins. Also, compounds from sponges have
proven effective against lipoxygenase-mediated conditions in humans
(See U.S. Pat. No. 6,750,247 to Crews et al.)
[0004] Tunicates have proven to be an important source of bioactive
natural products. Among marine natural products that have advanced
as cancer treatments the ecteinascidins and didemnins are derived
from tunicates, and the eudistomins have potent antiviral activity.
As part of an ongoing study of bioactivity among Antarctic marine
invertebrates, the inventors had the occasion to study the tunicate
Synoicum adareanum.
[0005] S. adareanum is a circumpolar tunicate common in the shallow
waters around Anvers island (64.degree. 46'S, 64.degree. 03'W) on
the Antarctic Peninsula from 15 to 796 meters depth. S. adareanum
colonies consist of large rounded or club-shaped heads with the
bottom stalk being wrinkled and leathery and only slightly narrower
than the head. S. adareanum colonies can be up to eighteen
centimeters high with a diameter of twelve centimeters. S.
adareanum colonies may comprise a single head or, up to six heads
can arise from a single stalk.
SUMMARY OF INVENTION
[0006] Extracts from S. adareanum, Palmerolide A (1), Palmerolide
C, Palmerolide D, and Palmerolide E displayed bioactivity in
field-based feeding-deterrent assays, leading the inventors to
investigate the chemical nature of the activity. Presented are
novel, isolated polyketides, Palmerolide A (1), Palmerolide C,
Palmerolide D, and Palmerolide E as the major natural product from
extracts of S. adareanum. These polyketides display selective
cytotoxicity in the National Cancer Institute (NCI) 60 cell line
panel inhibiting, inter alia, melanoma (UACC-64, LC.sub.50 0.018
.mu.M) with three orders of magnitude greater sensitivity relative
to other cell lines tested.
[0007] In a general embodiment, the present invention provides a
method of treating a subject with cancer, comprising administering
to the subject a therapeutically effective amount of at least one
isolated compound obtained from extracts of a Synoicum species. In
this embodiment, the Synoicum species is S. adareanum and the
isolated compound obtained from the Synoicum species is a
Palmerolide. The Palmerolide is chosen from the group consisting of
Palmerolide A(1), Palmerolide C, Palmerolide D, and Palmerolide
E.
[0008] In an alternate embodiment, a composition (or an isomer,
racemate or racemic mixture thereof, or a pharmaceutically
acceptable salt thereof) is provided comprising an isolated
compound of the formula:
##STR00001##
[0009] In yet another embodiment the present invention provides for
a composition (or an isomer, racemate or racemic mixture thereof,
or a pharmaceutically acceptable salt thereof) comprising an
isolated compound of the formula:
##STR00002##
[0010] An additional embodiment the present invention provides for
a composition (or an isomer, racemate or racemic mixture thereof,
or a pharmaceutically acceptable salt thereof) comprising an
isolated compound of the formula:
##STR00003##
[0011] The present invention also provides for a composition (or an
isomer, racemate or racemic mixture thereof, or a pharmaceutically
acceptable salt thereof) comprising an isolated compound of the
formula:
##STR00004##
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a perspective view of the chemical formula for
Palmerolide A.
[0013] FIG. 2 is a chart showing the NMR Data for Palmerolide
A.
[0014] FIG. 3 depicts selected ROE correlations relating the
relative stereochemistry between C-11 and C-19.
[0015] FIG. 4 is a chart showing the National Cancer Institute
(NCI) Developmental Therapeutics Program In-Vitro Testing Results
for Palmerolide A.
[0016] FIG. 5 is a continued chart, showing the National Cancer
Institute (NCI) Developmental Therapeutics Program In-Vitro Testing
Results for Palmerolide A.
[0017] FIG. 6 is a graph showing National Cancer Institute (NCI)
Developmental Therapeutics Program Dose Response Curves for all
cell lines tested for Palmerolide A.
[0018] FIG. 7 is a graph showing National Cancer Institute (NCI)
Developmental Therapeutics Program Dose Response Curves for
Melanoma cell lines tested for Palmerolide A.
[0019] FIG. 8 is a graph showing National Cancer Institute (NCI)
Developmental Therapeutics Program Dose Response Curves for Colon
Cancer cell lines tested for Palmerolide A.
[0020] FIG. 9 is a graph showing National Cancer Institute (NCI)
Developmental Therapeutics Program Dose Response Curves for Renal
Cancer cell lines tested for Palmerolide A.
[0021] FIG. 10 is perspective view of the chemical formula for
Palmerolide C.
[0022] FIG. 11 is a chart showing the NMR Data for Palmerolide
C.
[0023] FIG. 12 is a chart showing the National Cancer Institute
(NCI) Developmental Therapeutics Program In-Vitro Testing Results
for Palmerolide C.
[0024] FIG. 13 is a continued chart, showing the National Cancer
Institute (NCI) Developmental Therapeutics Program In-Vitro Testing
Results for Palmerolide C.
[0025] FIG. 14 is a graph showing National Cancer Institute (NCI)
Developmental Therapeutics Program Dose Response Curves for all
cell lines tested for Palmerolide C.
[0026] FIG. 15 is a perspective view of the chemical formula for
Palmerolide D.
[0027] FIG. 16 is a chart showing the NMR Data for Palmerolide
D.
[0028] FIG. 17 is a perspective view of the chemical formula for
Palmerolide E.
[0029] FIG. 18 is a chart showing the NMR Data for Palmerolide
E.
[0030] FIG. 19 is a chart showing the National Cancer Institute
(NCI) Developmental Therapeutics Program In-Vitro Testing Results
for Palmerolide E.
[0031] FIG. 20 is a continued chart, showing the National Cancer
Institute (NCI) Developmental Therapeutics Program In-Vitro Testing
Results for Palmerolide E.
[0032] FIG. 21 is a graph showing National Cancer Institute (NCI)
Developmental Therapeutics Program Dose Response Curves for all
cell lines tested for Palmerolide E.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0033] In the following detailed description of the preferred
embodiments, reference is made to the accompanying drawings, which
form a part hereof, and within which are shown by way of
illustration specific embodiments by which the invention may be
practiced. It is to be understood that other embodiments may be
utilized and structural changes may be made without departing from
the scope of the invention.
Terms
[0034] Those skilled in the art will recognize that the Palmerolide
compounds disclosed herein can exist in several tautomeric forms.
All such tautomeric forms are considered as part of this
invention.
[0035] "Pharmaceutically acceptable carrier" refers to any carrier,
diluent, excipient, wetting agent, buffering agent, suspending
agent, lubricating agent, adjuvant, vehicle, delivery system,
emulsifier, disintegrant, absorbent, preservative, surfactant,
colorant, flavorant, or sweetener, preferably non-toxic, that would
be suitable for use in a pharmaceutical composition.
[0036] "Pharmaceutically acceptable equivalent" includes, without
limitation, pharmaceutically acceptable salts, hydrates,
metabolites, prodrugs and isosteres. Many pharmaceutically
acceptable equivalents are expected to have the same or similar in
vitro or in vivo activity as the compounds of the invention.
[0037] "Pharmaceutically acceptable salt" refers to a salt of the
inventive compounds which possesses the desired pharmacological
activity and which is neither biologically nor otherwise
undesirable. The salt can be formed with acids that include,
without limitation, acetate, adipate, alginate, aspartate,
benzoate, benzenesulfonate, bisulfate butyrate, citrate,
camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,
glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride
hydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate,
maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
oxalate, thiocyanate, tosylate and undecanoate. Examples of a base
salt include ammonium salts, alkali metal salts such as sodium and
potassium salts, alkaline earth metal salts such as calcium and
magnesium salts, salts with organic bases such as dicyclohexylamine
salts, N-methyl-D-glucamine, and salts with amino acids such as
arginine and lysine. The basic nitrogen-containing groups can be
quarternized with agents including lower alkyl halides such as
methyl, ethyl, propyl and butyl chlorides, bromides and iodides;
dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl
sulfates; long chain halides such as decyl, lauryl, myristyl and
stearyl chlorides, bromides and iodides; and aralkyl halides such
as benzyl and phenethyl bromides.
[0038] "Prodrug" refers to a derivative of the inventive compounds
that undergoes biotransformation, such as by metabolism, before
exhibiting a pharmacological effect.
[0039] The prodrug is formulated with the objective of improved
chemical stability, improved patient acceptance and compliance,
improved bioavailability, prolonged duration of action, improved
organ selectivity, improved formulation (e.g., increased
hydrosolubility), and/or decreased side effects (e.g., toxicity).
The prodrug can be readily prepared from the inventive compounds
using methods known in the art, such as those described by Burger's
Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp.
172-178, 949-982 (1995).
[0040] "Palmerolide," as used herein, refers to a multi-membered
macrocyclic polyketide bearing carbonate and amide functionality.
In one embodiment, the Palmerolide is isolated from the tunicate
Synoicum adareanum; collected from the vicinity of Palmer Station
on the Antarctic Peninsula.
[0041] "Polyketides," as used herein, refers to any natural
compound containing alternating carbonyl and methylene groups
.beta.-polyketones), derived from repeated condensation of acetyl
coenzyme A.
[0042] "Macrocycle," as used herein, refers to a large molecule
arranged in a circle with various semi-compounds attached at
various points. The point of attachment and the nature of the
sub-molecule determines the nature and physiological effect of the
compound which contains it.
[0043] "Macrolide," as used herein, refers to a class of
antibiotics characterized by molecules made up of large-ring
lactones.
[0044] "Olefin," as used herein, is synonymous with "alkene" and
refers to an acyclic hydrocarbon containing one or more double
bonds.
[0045] As used herein, "a clinical response" is the response of a
cell proliferative disorder, such as melanoma, colon and renal
cancer, to treatment with novel compounds disclosed herein.
Criteria for determining a response to therapy are widely accepted
and enable comparisons of the efficacy alternative treatments (see
Slapak and Kufe, Principles of Cancer Therapy, in Harrisons's
Principles of Internal Medicine, 13.sup.th edition, eds.
Isselbacher et al., McGraw-Hill, Inc. 1994). A complete response
(or complete remission) is the disappearance of all detectable
malignant disease. A partial response is an approximately 50
percent decrease in the product of the greatest perpendicular
diameters of one or more lesions. There can be no increase in size
of any lesion or the appearance of new lesions. Progressive disease
means at least an approximately 25 percent increase in the product
of the greatest perpendicular diameter of one lesion or tumor or
the appearance of new lesions or tumors. The response to treatment
is evaluated after the subjects had completed therapy.
Pharmaceutical Compositions
[0046] A "pharmaceutical composition" of the invention is
formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, intravenous, intradermal, subcutaneous, oral
inhalation, transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0047] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, or phosphate buffered saline (PBS). In all
cases, the composition must be sterile and should be fluid to the
extent that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. Prevention of the
action of microorganisms can be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0048] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0049] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring. For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, a gas such as
carbon dioxide, or a nebulizer.
[0050] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art. The compounds can also be prepared in
the form of suppositories (for example, with conventional
suppository bases such as cocoa butter and other glycerides) or
retention enemas for rectal delivery.
[0051] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art.
[0052] A "therapeutically effective amount" is the amount of
Palmerolide A, C, D, or E, or any combination thereof necessary to
provide a therapeutically effective amount of the corresponding
compound in vivo. The amount of the compound must be effective to
achieve a response, such as, but not limited to total prevention of
(protection against) and to improved survival rate or more rapid
recovery, or improvement or elimination of symptoms associated with
a cellular proliferative disease or other indicators as are
selected as appropriate measures by those skilled in the art. In
accordance with the present invention, a suitable single dose size
is a dose that is capable of preventing or alleviating (reducing or
eliminating) a symptom in a patient when administered one or more
times over a suitable time period. One of skill in the art can
readily determine appropriate single dose sizes for systemic
administration based on the size of a mammal and the route of
administration.
Example I
Hollow Fiber Assay for Preliminary In Vivo Testing
[0053] The Biological Testing Branch of the Developmental
Therapeutics Program has adopted a preliminary in vivo screening
tool for assessing the potential anticancer activity of compounds
identified by the large scale in vitro cell screen. This hollow
fiber based assay has been in use since June, 1995.
[0054] Each compound is tested against a standard panel of 12 human
tumor cell lines including NCI-H23, NCI-H522, MDA-MB-231,
MDA-MB-435, SW-620 COLO 205, LOX IMVI, UACC-62, OVCAR-3, OVCAR 5,
U251 and SF-295. The cell lines are cultivated in RPMI-1640
containing 10% FBS and 2 mM glutamine. On the day preceding hollow
fiber preparation the cells are given a supplementation of fresh
medium to maintain log phase growth. For fiber preparation the
cells are harvested by standard trypsinization technique and
resuspended at the desired cell density (varies by cell line
between 2-10.times.10.sup.6 cells/ml). The cell suspension is
flushed into 1 mm I.D. polyvinylidene hollow fibers with a
molecular weight exclusion of 500,000 Da. The hollow fibers are
heat-sealed at 2 cm intervals and the samples generated from these
seals are placed into tissue culture medium and incubated at
37.degree. C. in 5% CO.sub.2 for 24-48 hours prior to implantation.
A total of 3 different tumor lines are prepared for each experiment
so that each mouse receives 3 intraperitoneal implants (1 of each
tumor line) and 3 subcutaneous implants (1 of each tumor line). On
the day of implantation, samples of each tumor cell line are
quantitated for viable cell mass by a stable endpoint MTT assay so
that the time zero (0) cell mass is known. Thus, the cytostatic and
cytocidal capacities of the test compound can be assessed. Mice are
treated with experimental agents starting on day 3 or 4 following
fiber implantation and continuing once daily for a total of 4
doses. Each agent is assessed by intraperitoneal injection at 2
dose levels with 3 mice/dose/experiment. Vehicle controls consist
of 6 mice receiving the compound diluent only. The fibers are
collected from the mice on the day following the fourth compound
treatment and subjected to the stable endpoint MTT assay. The
optical density of each sample is determined spectrophotometrically
at 540 nm and the mean of each treatment group is calculated. The
percent net cell growth in each treatment group is calculated and
compared to the percent net cell growth in the vehicle treated
controls. Each compound is assessed in a total of 4 experiments (3
cell lines/experiment.times. 4 experiments=12 cell lines).
[0055] Compounds are selected for further testing (e.g. time/dose
exposure studies preliminary pharmacology studies, subcutaneous
xenograft efficacy studies) on the basis of several hollow fiber
assay criteria. These include: (1) a reduction in net cell growth
of 50% or greater in 10 of the 48 possible test combinations (12
cell lines.times. 2 sites.times. 2 compound doses); (2) a reduction
in net cell growth of 50% or greater in a minimum of 4 of the 24
distant site combinations (intraperitoneal drug/subcutaneous
culture); and/or (3) cell kill of 1 or more cell lines in either
implant site (reduction in the viable cell mass below the level
present at the start of the experiment).
[0056] To simplify evaluation, a point system has been adopted
which allows rapid viewing of the activity of a given compound. For
this, a value of 2 is assigned for each compound dose which results
in a 50% or greater reduction in viable cell mass. The
intraperitoneal and subcutaneous samples are scored separately so
that criteria (1) and (2) can be evaluated. Compounds with a
combined IP+SC score 20, a SC score 8 or a net cell kill of one or
more cell lines can be considered for further studies. The maximum
possible score for an agent is 96 (12 cell lines.times. 2
sites.times. 2 dose levels.times. 2 [score]). These criteria were
statistically validated by comparing the activity outcomes of
>80 randomly selected compounds in the hollow fiber assay and in
xenograft testing. This comparison indicated that there was a very
low probability of missing a xenograft active compound if the
hollow fiber assay were used as the initial in viva screening tool.
Because of the design of the hollow fiber assay, the results of
individual cell lines are not reported since the statistical power
of the assay is based on the impact of a compound against the
entire panel of cells. In addition to the hollow fiber results,
other factors (e.g. unique structure, mechanism of action, etc.)
may result in referral of a compound for further studies without
the corn pound meeting these hollow fiber assay criteria.
Example II
Palmerolide Isolation
[0057] S. adareanum was extracted with 1:1 dichloromethane/methanol
and the residue resulting from rotary evaporation was partitioned
between an equal volume of water and ethyl acetate (EtOAc). Column
chromatography of the EtOAc partition fraction using mixtures of
hexane, ethyl acetate and methanol resulted in Fractions 4 and 5,
which eluted with 2%-5% methanol/ethyl acetate (310 mg) combined.
These combined fractions were further separated by gradient elution
of 1-10% MeOH/CHCl.sub.3 followed up by purification with HPLC on
C-18 (40% I-120/MeCN) afforded Palmerolide A, C, D and E (see Table
I below).
TABLE-US-00001 TABLE I ##STR00005##
Example III
[0058] As an illustrative example; Palmerolide A(1) was isolated as
a white amorphous solid from the 1:1 methanol/ethyl acetate
fraction eluting from silica gel chromatography of the crude (1:1
methanol/dichloromethane) extract. Mass spectrometric analysis
provided a molecular formula of C.sub.33H.sub.48N.sub.2O.sub.7
(FIG. 1) (HRFABMS m/z 585.3539, .DELTA. 0.1 mmu for [M.sup.++1]).
The C-1 to C-24 carbon backbone of Palmerolide A was unambiguously
established based on .sup.1H-.sup.1H and .sup.1H-.sup.13C
connectivity assignments from 2D NMR techniques as described
below.
[0059] The C-1 ester carbonyl of Palmerolide A (1) was found to be
conjugated to the C-2/C-3 olefin based on observation of
cross-correlations in the gHMBC spectrum (FIG. 2) from both H-2 and
H-3 to C-1. The olefinic protons were disposed trans based on the
large vicinal coupling (J=152 Hz). Three methylene carbons (.delta.
32.6, 25.7 and 38.5) were observed by both gCOSY and gHMBC to
intervene between the C-2/C-3 olefin and a hydroxymethine at
.delta. 3.83 (H-7). A trans-distributed olefin (J=15.5 Hz) could be
positioned between the aforementioned hydroxymethine and another at
.delta. 4.15 (H-10). While H-10 showed no gHMBC correlations, H-8,
H-9 and H-11 all displayed connectivity by gHMBC to C-10. H-11
could be further extended to C-12/C-13 (C-12 and C-13 are
coincident in the .sup.13C NMR spectrum) by gHMBC and gCOSY, as
well as to an ester carbonyl (OCOX) which displayed no further
connectivity using these NMR techniques. In the gHMBC spectrum,
H-13 coupled into the olefinic region, to C-14 and C-15. The
C-14/C-15 trans-olefin (J=14.6 Hz) was shown to be conjugated to a
tri-substituted olefin in positions C-16 and C-17 by gHMBC
correlations of H-14, H-15, H-18 and H-19, as well as H.sub.3-27.
The C-16/C-17 olefin must be E based on a ROESY spectrum, which
demonstrated the proximity (FIG. 3) of H.sub.3-27 to H-15. A
methylene group (C-18, .delta. 43.9) intervenes between the
C-16/C-17 olefin and an oxygen-bearing methine (C-19, .delta.
75.8), based on gHMBC correlations of H-16 and H.sub.3-27 to C-18;
H-19 and H-20 similarly correlate to C-18. The 20-membered
macrocycle was completed based on coupling between H-19 and the C-1
ester carbonyl in the gHMBC spectrum.
[0060] Features of the macrocycle were established by further
analysis of 2D NMR data. In addition to the four E olefins
described above, three oxygen atoms and one methyl group were
pendant on the macrocycle. Hydroxymethine protons at H-7 and H-10
were conclusively assigned based on observation of coupling of the
hydroxyl protons in both the gHMBC and gCOSY spectra: in the gHMBC
spectrum, the hydroxyl protons correlated to the respective x- and
.beta.-carbons, while in the gCOSY spectrum correlations were
observed between the hydroxyl protons and their respective
hydroxymethines. The third oxygen-bearing carbon (C-11), as
described above, correlates with an ester carbonyl (OCOX) at
.delta. 157.3.
[0061] Also pendant on the macrocycle is the C-19 side chain. The
H-20 multiplet, correlating to C-19 (gHMBC), was shown by gCOSY to
be coupled to a methyl group (C-26, .delta. 0.90) and the terminus
of a conjugated diene system based on H-19 and H-20 gHMBC
correlations to olefinic C-21 (.delta. 130.5). Both the C21/C-22
and the C-23/C-24 olefins were determined to be E, based, in the
former case, on a ROESY correlation between H.sub.3-25 and H-20,
and in the latter case the basis of coupling (J=14.2 Hz).
Connectivity of the C-23/C-24 olefin could be established based on
gHMBC correlations of H-23 to C-21, C-22, C-24 and C-25. C-24
marked the terminus of the contiguous carbon chain and could be
shown to bear an --NH group due to gHMBC correlations of an amide
proton at .delta. 9.84 to carbons C-23, C-24 and the amide
carbonyl, C-1' (.delta. 163.9).
[0062] The isopentenoyl substructure (C-1' to C-5') was unusual in
displaying .sup.4J.sub.CH coupling in the gHMBC spectrum between
the amide carbonyl (C-1') and both vinyl methyl groups (C-4' and
C-5'). Only one vinyl methyl can be placed within the
.sup.3J.sub.CH reach of the typical HMBC experiment optimized for 8
Hz. The 2-methyl-2-butenoyl isomer, wherein protons from one vinyl
methyl reside three bonds from the carbonyl and those from the
second reside four bonds distant was unlikely on chemical shift
grounds, but also because the vinyl methyl groups were mutually
correlated in the gHMBC spectrum, FIG. 2. The substructure was
secured as the isopentenoyl group by observation of very small
coupling (J=1.0 Hz) of the vinyl proton (H-2') to both vinyl methyl
groups (C-4' and C-5'), excluding a vicinal relationship (i.e.,
large J) between the vinyl proton and one vinyl methyl required by
the 2-methyl-2-butenoyl isomer.
[0063] The connectivity described above established the full planar
structure of Palmerolide A (1) with the exception of a single open
valence on the ester carbonyl attached to the macrolide at C-11.
Remaining to be accounted from the molecular formula was
--NH.sub.2. That the C-11 functional group was a carbamate is
supported by the precedence of that functional group on other
polyketides, most notably the anticancer agent discodermolide.
[0064] The stereochemical assignment of Palmerolide A's (1) five
asymmetric centers was established by the application of the
modified Mosher and Murata methods. (R)- and (S)-MTPA esters's of
Palmerolide A demonstrated both C-7 and C-10 to bear the R
configuration. Configurational analysis of the C-10/C-11 fragment
identified a gauche relationship between H-10 and H-11, based on
the small .sup.3J.sub.H-10/H-11 observed between the vicinal
protons and the large .sup.3J.sub.CH for both the H-10/C-12 and the
H-11/C-9 relationships. Further support for the conformation was
found in .sup.2J.sub.C-11/H-10 and .sup.2J.sub.C-10/H-11, both of
which were large and negative, defining the absolute
stereochemistry of C-11 as R. Similarly, configurational analysis
of the C-19/C-20 system suggested an anti relationship of the
respective protons, based on the large .sup.3J.sub.H-19/H-20, small
.sup.3J.sub.C-21/II-19, .sup.3J.sub.C-26/H-19 and
.sup.3J.sub.C-18/H-20, as well as the large .sup.3J.sub.C-19/H-20.
The relative position of C-18 in this fragment was secured by the
observation of ROESY correlations between H.sub.2-18 and H-20 as
well as H.sub.2-18 and H.sub.3-26 while no ROESY correlation was
observed between H.sub.2-18 and H-21, requiring the relative
configuration 19R*, 20S*.
[0065] The four olefins in the macrocycle constrain the flexibility
often found in macrolides, facilitating stereochemical analysis by
NOE studies. Further analysis of the ROESY spectrum revealed the
macrolide to adopt two largely planar sides of a tear-drop shaped
cycle, one side consisting of C-1 through C-6, the other C-11
through C-19, with C-7 through C-10 providing a curvilinear
connection. In particular, H-19, H.sub.3-27, H-15 and H.sub.2-13
(see FIG. 3) are sequentially correlated in the ROESY spectrum, as
are H.sub.3-26, H.sub.2-18, H-16. H-14 and H-12, defining the
periphery of the top and bottom face of the western hemisphere.
H-11 correlates only to the top series of protons, a result
consistent only with C-19 and C-11 both adopting the R
configuration. The absolute stereochemistry of the C-19/C-20
fragment is therefore 19R, 20S.
[0066] Tunicates are not well known as producers of type I
polyketides, though the patellazoles and iejimalides are
significant, bioactive, representatives. Palmerolide A (1) is
unusual in bearing a small macrocycle, with 20 members, compared to
24 in the patellazoles and iejimalides, and a vinyl amide, a
feature more commonly associated with cyanophyte-derived macrolides
such as tolytoxin. Palmerolide A displays cytotoxicity toward
several other melanoma cell lines. FIG. 2, [M14(LC.sub.500.076
.mu.M), SK-MEL-5 (6.8 .mu.M) and LOX IMVI (9.8 .mu.M)] as well as
the previously mentioned UACC-62. Besides melanoma, FIG. 3, one
colon cancer cell line (HCC-2998. 6.5 .mu.M), FIG. 4, and one renal
cancer cell line (RXF 393, 6.5 .mu.M), FIG. 5, Palmerolide A was
largely devoid of cytotoxicity (LC.sub.50>10 .mu.M),
representing a selectivity index among tested cell lines of
10.sup.3 for the most sensitive cells. Significantly, Palmerolide A
is COMPARE.-negative against the NCI database, suggestive of a
previously un-described mechanism of action. Field and laboratory
bioassay and chemical studies to address Palmerolide A's potential
are ongoing.
[0067] FIGS. 4 and 5, indicate the National Cancer Institutes
Developmental Therapeutics Program In-Vitro Testing Results for
Palmerolide A. FIG. 6 shows the National Cancer Institute (NCI)
Developmental Therapeutics Program Dose Response Curves for all
cell lines tested for Palmerolide A. In comparison, individual
results are shown for Melanoma (FIG. 7), Colon Cancer (FIG. 8) and
Renal Cancer (FIG. 9).
Example IV
Cytotoxicity of Palmerolide C
[0068] Palmerolide C, shown below and in FIG. 10, has the chemical
formula C.sub.33H.sub.49N.sub.2O.sub.7 (for NMR data see FIG. 11).
NCI cytotoxicity is shown in FIG. 12 and FIG. 13. NCI Dose Response
Curves for all cell lines are presented in FIG. 14.
##STR00006##
Example V
Cytotoxicity of Palmerolide D
[0069] Palmerolide D, shown below and in FIG. 15, has the chemical
formula C.sub.36H.sub.53N.sub.2O.sub.7. Palmerolide D NMR Data is
shown in FIG. 16.
##STR00007##
Example VI
Cytotoxicity of Palmerolide
[0070] Palmerolide E, shown below and in FIG. 17, has the chemical
formula C.sub.27H.sub.39NO.sub.7 (for NMR data see FIG. 18). NCI
cytotoxicity is shown in FIG. 19 and FIG. 20. NCI Dose Response
Curves for all cell lines are presented in FIG. 21.
##STR00008##
[0071] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
[0072] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
* * * * *